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DNA probe for enriching low-frequency DNA mutations and application of DNA probe

A DNA probe and low-frequency technology, applied in the direction of DNA/RNA fragments, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of high sample size and limit the application of simultaneous detection of multiple sites, and achieve Efficient enrichment, significant enrichment, and dynamic monitoring

Active Publication Date: 2019-01-08
杭州瑞普基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But they have a common disadvantage: only one mutation site can be detected at a time, and the requirement for sample size is very high, which limits its application for simultaneous detection of multiple sites

Method used

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  • DNA probe for enriching low-frequency DNA mutations and application of DNA probe
  • DNA probe for enriching low-frequency DNA mutations and application of DNA probe
  • DNA probe for enriching low-frequency DNA mutations and application of DNA probe

Examples

Experimental program
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Effect test

Embodiment 1

[0046] In this example, the L858R mutation on exon 21 of the EGFR gene hotspot mutation is taken as an example to analyze the method for capturing and enriching EGFR mutations by special DNA probes of the present invention. The samples used in the experiment were Horizon’s Multiplex I cfDNAReference Standard Set (HD780), which were EGFR gene wild type, with 5% EGFR L858R mutation, with 1% EGFR L858R mutation, and with 0.1% EGFR L858R mutation; 3 cases of lung adenocarcinoma Clinical samples.

[0047] The method for detecting the L858R mutation on exon 21 of the EGFR gene hotspot mutation by using the above method comprises the following steps:

[0048] (1) Separation of peripheral blood samples

[0049] 1. Collect one tube (10mL / tube) of the subject’s peripheral blood into a Streck tube, gently upside down (to prevent cell rupture) 6-8 times and mix well, and perform the following treatment within 7 days after blood collection;

[0050] 2. Centrifuge at 1600g for 10 minutes ...

Embodiment 2

[0109] In this example, the T790M mutation on exon 20 of the EGFR gene hotspot mutation is taken as an example to analyze the method for capturing and enriching EGFR mutations by special DNA probes of the present invention. The samples used in the experiment were Horizon’s Multiplex I cfDNAReference Standard Set (HD780), which were EGFR gene wild type, with 5% EGFR T790M mutation, with 1% EGFR T790M mutation, and with 0.1% EGFR T790M mutation; two cases of lung adenocarcinoma Clinical samples.

[0110] The method for detecting the T790M mutation on exon 20 of the EGFR gene hotspot mutation by using the above method comprises the following steps:

[0111] (1) Separation of peripheral blood samples

[0112] 1. Collect 1 tube (10mL / tube) of peripheral blood from the subject into a Streck tube, gently invert it upside down (to prevent cell rupture) 6-8 times and mix thoroughly, and perform the following treatment within 7 days after blood collection;

[0113] 2. Centrifuge at 16...

Embodiment 3

[0173] In this example, the deletion mutation on exon 19 of the EGFR gene hotspot mutation is taken as an example to analyze the method for capturing and enriching EGFR mutation with a special DNA probe of the present invention. The samples used in the experiment were Horizon’s Multiplex I cfDNAReference Standard Set (HD780), which were EGFR gene wild type, 5% EGFR 19DEL mutation, 1% EGFR 19DEL mutation, and 0.1% EGFR 19DEL mutation; two cases of lung adenocarcinoma Clinical samples.

[0174] The method for detecting the deletion mutation on exon 20 of EGFR gene hotspot mutation by using the above method comprises the following steps:

[0175] (1) Separation of peripheral blood samples

[0176] 1. Collect 1 tube (10mL / tube) of peripheral blood from the subject into a Streck tube, gently invert it upside down (to prevent cell rupture) 6-8 times and mix thoroughly, and perform the following treatment within 7 days after blood collection;

[0177] 2. Centrifuge at 1600g for 10 ...

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Abstract

The invention proposes a DNA probe for enriching low-frequency DNA mutations. The DNA probe is complementary to a sequence comprising the low-frequency DNA mutations and has an LNA modification. According to the fact that the probe provided by the embodiment of the invention is complementary to the sequence comprising the low-frequency DNA mutations, and the probe has the LNA modification; inventors found that when the probe is paired with the sequence comprising the low-frequency DNA mutations, if the low-frequency DNA mutations are present, the low-frequency DNA mutations are completely complementary to the base of the DNA probe, and a Tm value is higher than the Tm value of the base mismatch between a wild type DNA and the DNA probe and much more higher than that of the mismatch betweenthe low-frequency DNA mutations and a normal base without the LNA modification; therefore, the DNA probe binds more to the DNA containing the low-frequency mutations and less binds to the wild type DNA at a suitable temperature, and the low-frequency DNA mutations can be specifically enriched.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to DNA probes for enriching low-frequency DNA mutations and applications thereof, more specifically, the present invention relates to DNA probes for enriching low-frequency DNA mutations, probes in The use in the preparation of the kit, the kit, the method for enriching low-frequency DNA mutations and the method for detecting low-frequency DNA mutations. Background technique [0002] Lung cancer has a high morbidity and mortality rate. Its incidence rate is the second in the United States and the first in China, and its mortality rate is the first in both the United States and China. In addition to smokers, many people who have never smoked also suffer from lung cancer due to genetic and environmental factors. 85-90% of lung cancer patients are non-small cell lung cancer. After a lot of research in recent years, scientists have a clearer understan...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/6886C12Q1/6806
CPCC12Q1/6806C12Q1/6886C12Q2600/156C12Q2531/113
Inventor 林锐濮悦
Owner 杭州瑞普基因科技有限公司
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